Method and compositions for regulating plant growth



Feb. 28, 1956 F. c. STEWARD ETAL 2,735,644

METHOD AND COMPOSITIONS FOR REGULATING PLANT GROWTH Filed Dec. 5, 1954F19. Q BASAL MEDIUM '20 El BASAL MEDIUM c.M.

MG. FRESH WEIGHT OF CARROT CULTURES CONTROLS I0 I O.| GRAMS EQUIVALENTTISSUE PER IOO ML. MEDIUM Fig. 2

% EXTRACT FROM AERATED SLICES III EXTRACT FROMUNAERATED SLICES M I0 I .I.OI .OOI |5% C.M.+ EXTRACT OF POTATOIGRAMS POTATO PER.|OO

INVENTORS FREDERICK C. STEWARD 8: SAMUEL M. CAPLIN BY QMXAL ATTORNEY MG.FRESH WEIGHT OF CARROT CULTURES ML. MEDIUM) United States Patent METHODAND COMPOSITIONS FOR REGULATING PLANT GROWTH Frederick C. Steward,Ithaca, and Samuel M. Caplin, Rochester, N. Y.

Application December 3, 1954, Serial No. 473,007 6 Claims. (Cl. 71-2.3)

This invention relates to compositions comprising coconut milk and anhydroxylic extract of tissue of an angiosperm of the class consisting ofonion bulb and potato tuber. More particularly, this invention relatesto compositions comprising coconut milk factor in admixture with agrowth inhibitor for said coconut milk, the inhibitor being anhydroxylic extract of onion bulb tissue or of potato tissue.

This application is a continuation-in-part of our copending application,Serial No. 285,412, filed April 13, 1952, now abandoned.

Onion juice has been claimed to enhance the physiological activity ofauxins on plants. The growth-promoting activity both of indoleaceticacid and 2,4-D are reportedly increased by use of onion juice but thishas been attributed to its sugar content.

In our copending application, Serial No. 285,411, now abandoned, butfiled concurrently with our parent application, Serial No. 285,412, wehave described the synergistic effect of compositions containing coconutmilk and synthetic plant regulants such as 2,4-D on the promotion ofgrowth of potato tissue.

To our surprise we have now found that upon substituting onion extractfor 2,4-D in a nutrient medium containing coconut milk the growth ofcertain plant tis sue, such as for instance carrot root and Jerusalemartichoke tuber, is substantially inhibited. We have further found thatwhen an hydroxylic extract of tissue of another angiosperm, namely,potato tissue (widely used in microbiology as a general nutrient) isadmixed with a nutrient medium containing coconut milk the extractfunctions as does onion extract and inhibits the growth of plant cells.I

The invention will be described with particular reference to theinhibition of the growth promoting qualities of coconut milk asdetermined by the carrot assay method but it is to be understood thatthe compositions of our invention inhibit the growth of other types ofplant cells than those of the carrot root.

It is already known that the growth-promoting qualities of coconut milk,which enable rapid proliferative growth of the secondary phloem ofcarrot root to be obtained under tissue culture conditions, are due to aheat stable, water soluble substance or substances which are referred tohereinafter as the coconut milk growth factor (C. M. R). The isolationand identification of C. M. Rare more fully described and claimed in thecopending United States application of Frederick C. Steward and Edgar M.Shantz, Serial No. 365,182, filed June 30, 1953.

The growth factor can also be obtained from other sources notablyimmature corn (Zea) grains and other similar sources in which there is arelatively precocious growth of the endosperm.

The coconut milk factor, which is most conveniently found in the liquidendosperm of coconut, occurs at all stages after fertilization. In theimmature fruit of Zea it is especially abundant about 2 weeks afterpollination.

The factor is also found in the developing female gametophyte ofGinkgo-as a representative of a haploid nutritive tissue, and in certainplant tumors in which cells return to the permanently proliferatingstate.

In addition to coconut milk the compositions of this invention containan hydroxylie extract of an angiosperm of the class consisting of onionbulb and potato tuber. The inhibitor in potato is more abundant in theperiderm than in the parenchyma.

By the term, an hydroxylic extract, we mean to include aqueous extracts,lower aliphatic alcoholic (16 carbon atoms) extracts, and aqueous loweraliphatic alcoholic extracts.

The hydroxylic extracts of onion bulb and potato tissue are prepared byany conventional method. The angiosperm tissue in the fresh or driedstate is preferably extracted with hot water, although the inhibitorysubstances can also be extracted by other hydroxylic solvents such asfor instance, methyl alcohol, ethyl alcohol, npropyl alcohol, isobutylalcohol, isohexyl alcohol, and alcohol-water mixtures.

It will be understood that the living cells of the plant tissue arekilled by the hot water, or by the alcohol during the extraction step.In the event the tissue is autoclaved the living cells are also killed,the soluble constitucnts thereupon diffusing out.

The resulting extract may be filtered. The filtrate may be furtherdiluted preparatory to use in the compositions of our invention.

in operating in accordance with the present invention any suitableamount of the aforementioned plant extract may be used to inhibit thegrowth-promoting qualities of coconut milk on plant cells in anyconventional nutrient medium, such as for instance, Whites nutrientsolution (A Handbook of Plant Tissue Culture by Philip R. White. TheJacques Cattell Press, Lancaster, Pa. 1943).

The relative proportions of a given extract with coconut milk will varydepending upon such factors as the particular extract employed, theplant material whose growth is to be inhibited, the physiologicalcondition of the material so treated, and the environmental conditions.

It is impossible, therefore, to state exactly the proportions that willbe used in all situations. In general, the compositions of the inventionwill contain inhibitory substances in an extract of from about 1.0 to 10grams of plant tissue per ml. of medium, said medium containing about atleast 3% but not more than about 15% by volume of coconut milk or itsequivalent of isolated C. M. F. factor.

It will be understood that the failure of'mature plant cells to grow inthe presence of a full nutrient containing compositions of the inventionis' due to the presence of inhibitors for the growth-promoting qualitiesof coconut milk and not to too much coconut milk growth factor.

The utility of the present invention resides in the control that itgives over the ability of plant cells to grow by division: the growthbeing promoted by one set of factors (as in the use of coconut milk) andrepressed by another (as in the use of extracts of onion and potatotissue with the claimed inhibitory actions).

The nature of this invention will be better understood by reference tothe following illustrative examples:

Example 1 The inhibitory effect of a composition containing coconut milkand onion extract on the growth of carrot explants in a basal medium isillustrated by this example.

A description of the nutrient and other conditions under which standardexplants of carrot roots can be accurately and reproducibly grown isfound in our articles appearing in Nature 163,920 (1949) and Ann. Bot.N. S. 16, Nos. 62 and 64 (1952). It will be understood that one of thestandard conditions described in the aforementioned articles for .thegrowth of carrot-root ex plants is the use of a basal mediumsupplemented by 15 percent by volurneof coconut milk.

In brief, 'our carrot assay method makes use .of Pyrex glass:cultureitubes rounded at each end and mounted .on disks which areslowly revolved about ahorizontal shaft so that each tube is tumbled endover end. At the middle of each .tube vis a side neck through which thetransfer of cultures :can be made and, when plugged with cotton, throughwhich gas exchange can occur.

The cultures in these klinostat tubes were grown on a basal nutrientmedium having the following composition:

Salts: Mg./ liter 'Mgsoi 360.0 Ca(NO3)2 200.0 Na2SO4 200.0

KNO: 80.0 KCl '65 .0

NaHz'POeI-IZO 16.5

Fe2.(C4H4Oe')3 2.5 MnSO4 4.5

ZnSO4 1.5 H3BO3 1.5

Kl 0.75 Sucrose 20,0000 Glycine 3 .0 Nicotinic acid 0.5

Pyridoxine 0.1 Thiamin 0.1

All of the explants were obtained from carrot roots in the form ofcylinders, removed by a cannula, and cut with a multibladed cutter into3 mg. radial segments. Only phloem cylinders with their inner tangentialsurface 1 mm. from the cambium were used.

Hot water (about 50-80 C.') extracts of onion bulb tissue were made andadded to the nutrient medium for carrot explants. The data shown inFigure 1 are recorded in terms of the effect of onion juice on thegrowth of carrot explants under the above described standard conditions.

Example 2 The inhibitory effect of an aqueous potato tissue extract onthe growth of carrots in a culture containing coconut milk was asfollows:

Using the cannula used to cut carrot plugs, cylinders of potato tuberswere removed and placed in the rotating culture tubes together with.basal medium and coconut milk. The medium containing the tissue wasthen autoclavecl and the tube was inoculated with a standard carrotexplant.

.The effect of added potato extract .on the growth of .carrotin thepresence ofcoconut milk is shown in Figure We claim:

1. A plant growth regulating composition comprising coconut milk and, inan amount suflicient to exert inhibitory action on the plantgrowth-promoting activity of coconut milk, a hydroxylic extract of planttissue of an angiosperm of the class consisting of onion bulb and potatotuber, the extraction of from about 1 to 10 grams of said plant tissueyielding 'an extract containingan amount of an inhibitory substancesuificient to inhibit the plant growth-promoting.activity of coconutmilk when said extract is mixed with 1100 milliliters of nutrient mediumcontaining from about 3 to 15% by'volume of coconut milk.

2. A composition of claim 1 wherein the extract is an aqueous extract.

3. A composition of claim 1 wherein the extract is an alcoholic extract.

4. A plant growth .regulating composition comprising coconut milk and,in an .amount suflicient toexert inhibitor-y action on.coconutmilk-stimulated growth of .plant cells, an aqueous extract :of.onion bulbitissue, the aqueous extraction of from about 11 to 10 gramsof said tissue yielding an extract containingan amount of .an inhibitorysubstance 'sufiicient to inhibit the growth promoting .activity ofcoconut milk on plant cells in 100 milliliters of nutrient mediumcontaining from about 3 to 15% by volume of coconut milk.

5. A plant growth regulating composition comprising coconut milk and, inan :amount suflicient to exert'inhibitory action 'an coconutmilk-stimulated growth of plant cells,.anaqueous-extract.of-potatotissue, the aqueous .extraction of from about 1 to 10 grams of saidtissue yielding an amount of inhibitory substance suflicient to inhibitthe growth-promoting activityof coconut milk .on plant cells in 100milliliters of nutrient medium containing from about 31to 15% byvolumeof coconut milk.

6. A method which comprises applying to plant tissue aeompositioncontaining coconut milk and, in an amount sufiicient to exertinhibitory action on the growth promoting activity of coconutmilk,an'hydroxylic extract of plant tissue of :an angiosperm selected fromthe group consisting of onion and potato, the extraction of from about-1 to 10 grams of said plant tissue yielding an extractcontaining anamount of an inhibitory substance sufiicient'to inhibit thegrowth-promoting activity of coconut milk in 100 milliliters'of nutrientmedium containing from about '3 to 15% by volume of coconut milk.

References Cited'inthe fileof this .patent Science, vol. 108, Dec. 10,1948, pages'6'5'5 to 657.

Cosmopolitan Fish Cookery for the Phillipines,'U. S. "Dept. of'theInterior, Fish and Wildlife Service Avery 'Leaflet 377 .(Recd. in U. S.Patent Ofiice Libray August 18, 1950), pages'2'8 and .129.

1. A PLANT GROWTH REGULATING COMPOSITION COMPRISING COCONUT MILK AND, IN AN AMOUNT SUFFICIENT TO EXERT INHIBITORY ACTION ON THE PLANT GROWTH-PROMOTING ACTIVITY OF COCONUT MILK, A HYDROXYLIC EXTRACT OF PLANT TISSUE OF AN ANGIOSPERM OF THE CLASS CONSISTING OF ONION BULB AND POTATO TUBER, THE EXTRACTION OF FROM ABOUT 1 TO 10 GRAMS OF SAID PLANT TISSUE YIELDING AN EXTRACT CONTAINING AN AMOUNT OF AN INHIBITORY SUBSTANCE SUFFICIENT TO INHIBIT THE PLANT GROWTH-PROMOTING ACTIVITY OF COCONUT MILK WHEN SAID EXTRACT IS MIXED WITH 100 MILLILITERS OF NUTRIENT MEDIUM CONTAINING FROM ABOUT 3 TO 15% BY VOLUME OF COCONUT MILK. 